Gonadal Receptors/mechanisms Of Action--Peptide Hormones

性腺受体/作用机制——肽激素

• 批准号: 6534877
• 负责人: MARIA DUFAU
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6534877
• 关键词: animal genetic material tag; cell growth regulation; chorionic gonadotropin; gene targeting; genetic mapping; genetic promoter element; genetic transcription; genetically modified animals; gonadotropins; gonads; hormone receptor; hormone regulation /control mechanism; human genetic material tag; human tissue; laboratory mouse; laboratory rat; luteinizing hormone; peptide hormone; posttranslational modifications; prolactin; protein structure function; receptor binding; receptor expression; steroid biosynthesis; tissue /cell culture

1) The LH receptor: The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that plays an essential role in gonadal development and differentiation. Our previous studies demonstrated regulation of the Sp1/Sp3-driven TATAless promoter of the human LH receptor by the orphan receptors EAR2 and EAR3/COUP-TF1 (inhibitory) and TR4 (stimulatory). These orphans receptors bound competitively and with high affinity to an imperfect direct-repeat motif composed of a estrogen response element half-site and a second degenerate half-site (DR). Current studies investigated the differential binding of orphan receptors to rat and human LHR promoters, and their modulation of LHR transcription in rat granulosa cells differentiated in culture by hormone treatment. This process resembles the induction of the LHR gene in granulosa cells of the human ovary and permits analysis of the role of orphan receptors during gonadal cell differentiation from early stages to luteinization. These studies demonstrated repression of rat LHR gene transcription by EAR2 and EAR3/COUP-TFI through their binding to a DR domain (albeit with 2-fold lower affinity than the human). No binding or activation was found for TR4 due to a single nucleotide difference at the second half-site of the DR. The lower binding affinity for EAR2 and EAR3 in the rat was associated with a smaller inhibitory effect than that observed in the human and was attributable to species differences in the adjacent 3-prime sequences. hCG treatment markedly reduced the inhibition of the rat LHR in granulosa cells and also decreased EAR2 and EAR3 protein levels. Abolition of the orphan receptor-mediated inhibition of the rLHR upon hCG treatment via derepression may contribute to the elevated LHR expression required for progression of granulosa cell maturation. 2) Gonadotropin regulation: Treatment with high doses of gonadotropins causes LHR-independent negative regulation of steroidogenic enzymes (steroidogenic desensitization) and up-regulation of a novel gonadotropin regulated RNA-helicase (GRTH). A previously unidentified protein that is constitutively present in Leydig cells and down-regulated by gonadotropin was recently cloned and characterized as a gonadotropin-regulated long chain acyl CoA synthetase (GR-LACS). The 79-kDa cytoplasmic protein is expressed in the pubertal and adult Leydig cells of the rat testis and shares sequence identity with two conserved regions of the LACS and luciferase families, but displays low overall amino acid similarities (23-28%). The expressed protein present in the cytoplasm of transfected cells displayed acyl CoA synthetase activity for long chain fatty acid substrates. In addition to its potential contributions to energy production and testicular steroidogenesis, GR-LACS could provide long chain acyl-CoA esters with regulatory effects on enzyme activity, membrane function, and gene expression. 3) Prolactin Receptors: Previous studies in this laboratory have mapped and resolved the genomic structure of the human prolactin gene of over 100 Kb . The studies demonstrated that the gene has a complex structure and is amenable to alternative splicing, and reported the presence of 10 exons (multiple non-coding Exons 1 and the common non-coding exon-2 and exon 3-10 coding for the long form of the receptor). In addition, a novel exon-11 of the human prolactin receptor was recently found to be distinct from its rodent counterpart, and two novel forms of the human prolactin receptor (S1a and S1b, which are derived from alternative splicing of exons 10 and 11) were identified. These new forms of the human prolactin receptor resemble the conventional receptor in having similar extracellular and transmembrane domain, but differ in having unique truncated intracellular domains. These short forms, which were found in several normal tissues and in breast cancer cell lines, are expressed as cell surface receptors and possess binding affinities comparable to the long form. However, unlike the long form, neither of the short forms mediates the prolactin induced-activation of the beta-casein gene promoter that is exhibited by the long form of the receptor. In contrast, these forms act as dominant negative repressors of the function of the long and intermediate receptor isoforms that mediated cell growth responses in human breast cancer cells. Due to the marked difference in the cellular levels of the two forms, which results from the more rapid turnover of S1a, the S1b form was a more effective inhibitor. These short forms with unique C-termini may exhibit distinct signaling pathways, in addition to modulating signaling from the long form of the receptor. These new receptors may have important roles in the diversified actions of prolactin in human tissues, and are of potential therapeutic relevance to the control of mammary cancer cell growth and immunoregulation. Other studies have addressed the consequences of disruption of the growth hormone receptor gene on testicular function. This work has provided an in vivo demonstration in GH receptor knockout mice that LH action on testosterone secretion is significantly impaired, due to a decrease in the number of testicular LH receptors. This is accompanied by diminished responsiveness of testicular steroidogenesis and decreased ability to convert androstenedione to testosterone. These changes are probably attributable to the absence of circulating IGF-I in the GH-deficient mice. These studies have indicated that IGF-I has a major role in the regulation of testicular endocrine function.

1)黄体生成素受体：黄体生成素受体(LHR)是一种G蛋白偶联受体，在性腺发育和分化中起重要作用。我们以前的研究表明，孤儿受体EAR2和EAR3/COUP-TF1(抑制性)和TR4(刺激性)调节Sp1/Sp3驱动的人促黄体生成素受体的TATAless启动子。这些孤儿受体竞争性地与一个不完善的直接重复基序结合，并具有很高的亲和力，该基序由雌激素反应元件的半个位点和第二个退化的半个位点(DR)组成。目前研究了孤儿受体与大鼠和人LHR启动子的差异结合，以及它们对激素诱导分化的大鼠颗粒细胞LHR转录的调控。这一过程类似于LHR基因在人类卵巢颗粒细胞中的诱导，并允许分析孤儿受体在性腺细胞从早期到黄体化分化过程中的作用。这些研究表明，EAR2和EAR3/COUP-TFI通过与DR结构域结合(尽管亲和力比人类低2倍)抑制了大鼠LHR基因的转录。大鼠对EAR2和EAR3的结合亲和力较低，其抑制作用比在人类中观察到的小，这是由于相邻3-碱基序列的物种差异所致。人绒毛膜促性腺激素能明显减轻对大鼠颗粒细胞LHR的抑制作用，并降低EAR2和EAR3蛋白水平。在hCG治疗中取消孤儿受体介导的抑制rLHR可能有助于颗粒细胞成熟所需的LHR表达的增加。2)促性腺激素调节：大剂量促性腺激素治疗引起LHR非依赖性类固醇生成酶的负调节(类固醇生成脱敏)和一种新的促性腺激素调节的RNA解旋酶(GRTH)的上调。最近克隆了一种先前未知的蛋白质，它存在于间质细胞中，受促性腺激素下调，并被鉴定为促性腺激素调节的长链酰基辅酶A合成酶(GR-LACS)。79 kDa的胞质蛋白在大鼠睾丸的青春期和成年期间质细胞中表达，并与LACS和荧光素酶家族的两个保守区序列相同，但总体氨基酸相似性较低(23%-28%)。表达于细胞质中的蛋白对长链脂肪酸底物具有酰基辅酶A合成酶活性。除了对能量产生和睾丸类固醇合成有潜在贡献外，GR-LACS还可以为长链酰基-辅酶A酯提供对酶活性、膜功能和基因表达的调节作用。3)催乳素受体：本实验室以前的研究已经绘制并解析了超过100kb的人催乳素基因的基因组结构。研究表明，该基因结构复杂，易于选择性剪接，并报道存在10个外显子(多个非编码外显子1和共同的非编码外显子-2和编码受体长形的外显子3-10)。此外，最近发现人催乳素受体的一个新的外显子-11与啮齿动物的外显子-11不同，并鉴定了两种新的人催乳素受体形式(S1a和S1b，它们来自外显子10和11的交替剪接)。这些新形式的人催乳素受体与传统的催乳素受体相似，具有相似的细胞外和跨膜结构域，但不同之处在于具有独特的截短的细胞内结构域。这些短形式在几个正常组织和乳腺癌细胞系中发现，以细胞表面受体的形式表达，并具有与长形式相当的结合亲和力。然而，与长形式受体不同的是，两种短形式受体都不介导催乳素诱导的β-酪蛋白基因启动子的激活，而长形式受体表现出这种激活。相反，这些形式是介导人乳腺癌细胞细胞生长反应的长和中间受体异构体功能的显性负抑制因子。由于S1a的更替速度较快，导致两种形式的细胞水平存在显著差异，S1b形式是一种更有效的抑制因子。这些具有独特C-末端的短形式除了调节来自长形式受体的信号外，还可能表现出不同的信号通路。这些新的受体可能在催乳素在人体组织中的多种作用中发挥重要作用，并对控制乳腺癌细胞生长和免疫调节具有潜在的治疗意义。其他研究已经解决了生长激素受体基因中断对睾丸功能的影响。这项工作在生长激素受体基因敲除的小鼠中提供了一个体内证明，由于睾丸黄体生成素受体数量的减少，黄体生成素对睾酮分泌的作用显著减弱。伴随而来的是睾丸类固醇合成反应减弱，雄烯二酮转化为睾酮的能力降低。这些变化可能是由于生长激素缺陷小鼠缺乏循环中的IGF-I所致。这些研究表明，IGF-I在睾丸内分泌功能的调节中起重要作用。